The long-term goal of this project is to identify and characterize genes that contribute to the development and progression of myelodysplastic syndromes (MDS). A better understanding of these genes may allow us to create effective therapies for patients with MDS and improve outcomes. An interstitial deletion involving one copy of the long arm of chromosome 5 (5q) is among the most common cytogenetic abnormalities in de novo and therapy-related MDS (25-40% of patients). We identified HSPA9 as a candidate gene located on the chromosome 5q31.2 commonly deleted region that contributes to altered hematopoiesis. We propose to define the mechanism of altered hematopoiesis caused by reduced levels of HSPA9 and to test whether Hspa9 haploinsufficiency contributes to MDS initiation and progression using primary human hematopoietic cells and heterozygous Hspa9 knock-out mice (Hspa9+/-), respectively, in the following specific aims: Specific Aim 1: We will determine whether the accelerated apoptosis and altered cell cycle kinetics observed following HSPA9 knockdown are p53-dependent. Because HSPA9 is known to physically interact with p53 in several models, we will determine whether they interact in primary human CD34+ cells. We will measure p53 activation in CD34+ cells following HSPA9 knockdown using a reporter assay and identify candidate p53-target genes responsible for the phenotype. We will validate the importance of p53 and the candidate genes using murine knockout models and RNA interference in primary human CD34+ cells. Specific Aim 2: We will establish whether haploinsufficiency of Hspa9 cooperates with Rps14 haploinsufficiency in mice. Mice lacking one copy of Rps14, a del(5q) gene, develop a macrocytic anemia and reduced progenitor cells. Most chromosome 5q deletions produce haploinsufficiency of both Hspa9 and Rps14 genes, suggesting that loss of both genes may cooperate in MDS. To directly test whether haploinsufficiency of Hspa9 and Rps14 cooperate during MDS initiation, we will intercross Hspa9+/- mice with mice lacking one copy of Rps14 and comprehensively characterize the hematopoiesis of singly or doubly heterozygous mice. Specific Aim 3: We will determine whether Hspa9 haploinsufficiency predisposes mice to develop leukemia. A devastating complication of MDS is the development of leukemia, which is highly-associated with deletions spanning chromosome 5q31.2. Identifying genes that contribute to del(5q31.2)-associated leukemia is vital to our understanding of this disease. To directly test whether haploinsufficiency of Hspa9 contributes to leukemia development, we will infect Hspa9+/- or wild-type littermate mice with a modified Moloney murine leukemia virus (MOL4070LTR retrovirus, a well established AML model) and monitor mice for the development of AML. We will identify and study candidate cooperating genes using mouse models.